Our project previously focused on the development of novel vaccine and therapeutic strategies using Yersinia pestis models. We examined lipid A mimetics (aminoalkyl glucosaminide phosphates, AGPs), Tolllike receptor (TLR) 4 agonists. We found that AGPs directly protect against pneumonic plague, dramatically augment antibiotic therapy, and are efficacious adjuvants for either intranasal (i.n.) or subcutaneous (s.c.) vaccines containing Y. pestis capsular (F1) and/or V-antigens. Our vaccination regimen results in a rapid and sustained acquired TH1 immune response which protects in mouse and rat models. We also are characterizing several newly identified Y. pestis virulence factors and their regulation. Significant advances have been made toward identifying additional virulence mechanisms by screening for attenuated strains using advanced bioinformatic analyses, comprehensive transposon libraries, and through animal and Caenorhabditis elegans models. Our specific aims in this proposal expand on these successes: Specific Aim 1: To further examine the transition between induction of innate immunity with TLR agonists and specific immunity directed against pneumonic plague. The product development of AGPs requires delineation of the differences between murine and human immune responses. Transgenic humanized TLR4/MD-2 mice will be used to assess responses to AGPs as therapeutics and as vaccine adjuvants. In addition we will examine combinations of various TLR agonists to increase protection. Specific Aim 2: Determine the role of regulatory genes and surface/extracellular proteins in Y. pestis infection and/or flea vector maintenance. We will systematically characterize regulatory mutations, cell surface/extracellular proteins, and glucose-regulated genes for their role in virulence with the ultimate goal of identifying novel targets for product development. Specific Aim 3: Identify natural mutations (pseudogenes) that are essential for Y. pestis pathogenesis. We will test the hypothesis that increased bacterial virulence is, in part, due to evolutionary loss of genetic information during adaptation to the host. We will use bioinformatics to identify the lost and/or inactivated functions in the genomes of Y. pestis strains which are functional in the closely related but less virulent Yersinia enteropathogens. These identified Y. pestis 'pseudogenes' will be repaired to determine if this restoration attenuates virulence. We refer to this process as 'reverse molecular Koch's postulates'.